Evaluation of Inoculum Preparation for Etest and EUCAST Broth Dilution to Detect Anidulafungin Polyresistance in Candida glabrata.

The influence of inoculum preparation in EUCAST broth dilution and Etest to detect the coexistence of resistant and susceptible Candida subpopulations (defined as polyresistance [PR]) was evaluated. Cocultures of two echinocandin-resistant and susceptible clinical C. glabrata strains were used to simulate the occurrence of mixed populations in clinical samples, and antifungal susceptibility testing was performed with standard and modified approaches of inoculum preparation. Polyresistant results manifested as microcolonies or double ellipses in Etest and in single reduced optical density (OD) values (dip in OD) in microdilution. The strict inclusion of five distinct colonies of 1:5 and 1:10 resistant and susceptible cocultures led to higher rates of PR and R results compared to including one to two colonies in inoculum preparation (30% and 26% for Etest and broth dilution, respectively). Modifying the inoculum preparation by increasing the turbidity from a 2 to a 4 McFarland standard before redilution to a 0.5 McFarland standard reliably enabled the detection of resistance, with better identification of PR by Etest than by broth dilution (82% versus 32%, respectively) and of resistant minimum inhibitory concentration (MIC) values in 18% of Etests and 67% of microdilutions. The highest identification of PR succeeded with Etest and a modified 3 McFarland standard approach of inoculum preparation. Our data demonstrate that inoculum preparation as recommended and practiced does not reliably identify resistant subpopulations in polyresistant Candida cultures. By increasing the inoculum size for Etest assays from a 2 to a 4 McFarland standard with subsequent redilution, we propose a simple adaptation to increase reliability.

Comparative evaluation of eight in vitro pharmacodynamic models of infection: Activity of moxifloxacin against Escherichia coli and Streptococcus pneumoniae as an exemplary example.

Use of pharmacodynamic in vitro models provides more clinically relevant information about the activities of antibiotics than static endpoints. Several models are used to simulate pharmacokinetics by dilution of the medium. It is discussed whether this procedure would result in a washout of bacteria, particularly if profiles with a short half-life are simulated. Methods have been developed to minimise the washout of bacteria. Bacteria are retained in the system either by centrifugation and resuspension, use of filters, a capillary unit, dialysis tubing or mathematical correction, versus systems with an unprotected outflow allowing a continuous washout of bacteria. None of these eight models has been directly compared with another. Therefore, an interlaboratory study was performed to address the question of whether or not washout matters. All laboratories used identical batches of media, bacteria, antibiotics and simulated pharmacokinetic profiles with a short or long half-life. Values of area under the bacterial kill-time curve (AUBKC), single-point kill rate and time to 3-log10 reduction of inoculum were calculated. These parameters did not differ significantly between the models. Differences were noted if the inoculum was prepared from the early logarithmic growth phase compared with the late logarithmic or stationary growth phase, resulting either in a pronounced or reduced antibacterial activity. Thus, preparation of inocula affects the results generated, whereas washout of bacteria has apparently a negligible impact on antibacterial activities.

Pre-incubation of ruminal inocula to assess in vitro gas production and digestibility / Pré-incubação de inóculo ruminal para avaliação da produção de gases e digestibilidade in vitro

ABSTRACT: In vitro gas production techniques represent a valuable tool to describe the kinetics of ruminal degradation of food. However, the ruminal liquor used as a microbial inoculum has been a great source of variation and error. A standardization of this factor should contribute to assure the independence of food fermentation parameters from those of the inocula. In this research it was hypothesized that a controlled pre-incubation treatment of ruminal liquor could contribute to stabilize and homogenize the undigested residues of blanks and as a consequence, of the production of residual cumulative gas production (CGP). A pre-incubation (i.e. previous real incubation) of rumen inocula was developed with a simple substrate similar to the diet offered to donors at 1% w/v for 0, 1, 2 and 4 h (Control, Prei-1, Prei-2 and Prei-4 treatments respectively). Once the pre-incubation hours were completed, they were incubated with contrasting substrates and without substrate (i.e. blanks) in order to evaluate the CGP, in vitro digestibility of the DM and fermentation products. Although, the fermentative activity of the pre-incubated inoculums worked satisfactorily in the in vitro system, contrary to what was speculated, residues of the pre-incubation increased the variability and heterogeneity of variances among blanks. Consequently, it was concluded that the pre-incubations did not work to generate more homogeneous and less variable ruminal liquor for the in vitro gas production system.

RESUMO: Técnicas de produção de gás in vitro representam uma ferramenta valiosa para descrever a cinética de degradação ruminal dos alimentos. No entanto, o líquido ruminal utilizado como inóculo microbiano tem sido uma grande fonte de variação e erro. A padronização deste fator deve contribuir para garantir a independência dos parâmetros de fermentação dos alimentos a partir dos inóculos. Neste trabalho, hipotetizou-se que um tratamento controlado de pré-incubação do líquido ruminal poderia contribuir para estabilizar e homogeneizar os resíduos não digeridos dos brancos e, como conseqüência, da produção de produção cumulativa de gás residual (CGP). Uma pré-incubação (ou seja, incubação real prévia) dos inóculos do rúmen foi desenvolvida com um substrato simples semelhante à dieta oferecida aos doadores a 1% p/v por 0, 1, 2 e 4 h (Controle, Prei-1, Pré- 2 e Prei-4 tratamentos respectivamente). Uma vez completadas as horas de pré-incubação, elas foram incubadas com substratos contrastantes e sem substrato (ou seja, brancos) para avaliar o CGP, a digestibilidade in vitro da MS e os produtos de fermentação. Embora a atividade fermentativa dos inóculos pré-incubados tenha funcionado satisfatoriamente no sistema in vitro, ao contrário do que foi especulado, os resíduos da pré-incubação aumentaram a variabilidade e heterogeneidade das variâncias entre os brancos. Consequentemente, concluiu-se que as pré-incubações não funcionaram para gerar um líquido ruminal mais homogêneo e menos variável para o sistema de produção de gás in vitro.

Design and Operation of a Pilot-Scale Packed-Bed Bioreactor for the Production of Enzymes by Solid-State Fermentation.

In this review, we describe our experience in building a pilot-scale packed-bed solid-state fermentation (SSF) bioreactor, with provision for intermittent mixing, and the use of this bioreactor to produce pectinases and lipases by filamentous fungi. We show that, at pilot scale, special attention must be given to several aspects that are not usually problematic when one works with laboratory-scale SSF bioreactors. For example, it can be a challenge to produce large amounts of inoculum if the fungus does not sporulate well. Likewise, at larger scales, the air preparation system needs as much attention as the bioreactor itself. Sampling can also be problematic if one wishes to avoid disrupting the bed structure. In the fermentations carried out in the pilot bioreactor, when the substrate bed contained predominantly wheat bran, the bed shrank away from the walls, providing preferential flow paths for the air and necessitating agitation of the bed. These problems were avoided by using beds with approximately 50% of sugarcane bagasse. We also show how a mathematical model that describes heat and water transfer in the bed can be a useful tool in developing appropriate control schemes. Graphical Abstract.

Determination of Menaquinone-7 by a Simplified Reversed Phase- HPLC Method.

BACKGROUND: An efficient and accurate HPLC method was developed for the determination of menaquinone-7 (MK-7) in microbial fermentation using 2-propanol and n-hexane as extraction solvents as well as the eluent. METHODS: Extraction was carried out with 2-propanol and n-hexane (2:1, v/v) after enzymatic hydrolysis with 1% (w/v) lipase and ethanol water treatment prior to quantification in order to remove interfering lipids and denatured proteins. Chromatographic separation of MK-7 was accomplished isocratically on a C 18 Gemini column using a mobile phase mixture of 2- propanol: n-hexane (2:1, v/v) with a flow rate of 0.5 mL/min. UV detection was carried out from 200-400 nm and the chromatogram was extracted at a wavelength of 248 nm. A linear response was shown by the method with a coefficient of determination (R2) value of 0.9982. RESULTS: The recoveries of MK-7 were greater than 94% and the intra and inter day R.S.D values were less than 2%, demonstrating the accuracy of the method. The lower limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 µg/mL and 0.29 µg/mL, respectively. CONCLUSION: The general usefulness of the described method is demonstrated by the application of this method in the analysis of MK-7 from Bacillus species. Under these conditions, the analysis of MK-7 was achieved in less than 8 minutes with a retention time of 7.19 ± 0.1 minutes.

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture.

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10-30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 10(9) BHK-21 cells from 4 × 10(6) cells in 13 day with 1,051 mL culture medium.

Evaluation of an optimal preparation of human standardized fecal inocula for in vitro fermentation studies.

This study investigated the optimal preservation approach to prepare human feces as inoculum for in vitro fermentations as an alternative to the use of fresh feces. The four treatments studied were: Treatment 1) fresh feces resuspended in dialysate solution+glycerol; Treatment 2) fresh feces resuspended in dialysate solution+glycerol and then stored at -80°C; Treatment 3) fecal sample frozen with 1.5 g glycerol; and Treatment 4) fecal sample frozen. All the treatments contained 8.75 g of feces, 3.5 ml dialysate and 4.9 ml glycerol when inoculated in TIM-2 in vitro system. Treatment 1 (fresh fecal preparation) was used as a reference. The effects were evaluated in terms of i) metabolic activity and ii) composition of the microbiota using fermentation experiments in the TIM-2 in vitro system. In all treatments, high levels of acetate were produced followed by n-butyrate and propionate. However, the metabolic activity of the bacteria, in terms of short-chain fatty acid production, was affected by the different treatments. Microbiota composition was analyzed using the IS-pro profiling technique. Diversity in Actinobacteria, Firmicutes, Fusobacteria and Verrucomicrobia and Proteobacteria groups seemed to be preserved in all treatments whereas it was observed to decline in the Bacteroidetes group. Preparing a human fecal inoculum resuspended in dialysate solution with glycerol and then stored at -80°C showed high similarities to the results obtained with fresh feces, and is proposed as the optimal way to freeze fecal material as an alternative to fresh feces for in vitro fermentation studies.

To pool or not to pool? Impact of the use of individual and pooled fecal samples for in vitro fermentation studies.

This study investigated the stability and the activity of the microbiota from a single and a pool of donors in the TNO in vitro model of the colon (TIM-2 system). Our findings demonstrate the suitability of the preparation of a pool of fecal sample to be used for fermentation experiments.

Xylitol production from wheat straw hemicellulosic hydrolysate: hydrolysate detoxification and carbon source used for inoculum preparation / Produção de xilitol em hidrolisado hemicelulósico de palha de trigo: destoxificação do hidrolisado e fonte de carbono utilizada para o preparo do inóculo

Wheat straw hemicellulosic hydrolysate was used for xylitol bioproduction. The use of a xylose-containing medium to grow the inoculum did not favor the production of xylitol in the hydrolysate, which was submitted to a previous detoxification treatment with 2.5 percent activated charcoal for optimized removal of inhibitory compounds.

Hidrolisado hemicelulósico de palha de trigo foi utilizado para a bioprodução de xilitol. O uso de meio contendo xilose para crescer o inóculo não favoreceu a produção de xilitol no hidrolisado, que foi submetido a um tratamento prévio de destoxificação com 2.5 por cento de carvão ativo para remoção otimizada de compostos inibitórios.

Xylitol production from wheat straw hemicellulosic hydrolysate: hydrolysate detoxification and carbon source used for inoculum preparation.

Wheat straw hemicellulosic hydrolysate was used for xylitol bioproduction. The use of a xylose-containing medium to grow the inoculum did not favor the production of xylitol in the hydrolysate, which was submitted to a previous detoxification treatment with 2.5% activated charcoal for optimized removal of inhibitory compounds.